human nrf2 sirna  (Santa Cruz Biotechnology)

Journal: iScience

Article Title: Inflammatory CD4 + T cells can waive NRF2-dependent SLC7A11-mediated cystine uptake by using ASCT1

doi: 10.1016/j.isci.2026.115680

Figure Lengend Snippet: Regulation of lipid peroxidation is extrinsically regulated by the SLC7A11 pathway in CD4 + T cells (A) Statistical analysis of SLC7A11 protein expression assessed by flow cytometry (shown as mean fluorescent intensity (MFI). CD4 + T cells were either left unstimulated (control) or measured at a distinct time point after stimulation with anti-CD3/CD28 antibodies. N = 3, 3 independently performed experiments, a one-way ANOVA multiple comparison test was performed. The calculated power of this experiment is 0.84. (B) ΔCT of SLC7A11 mRNA expression in human CD4 + T cells with and without anti-CD3/CD28 stimulation. N = 13, 4 independently performed experiments. (C) Gene expression in CD4 + T cells from WT and VAV cre Keap fl/fl (Keap1-KO) mice was assessed by microarray analysis. Colors indicate significant upregulation (red), or downregulation (green) compared to WT. (D) ΔCT of SLC7A11 mRNA expression in CD4 + T cells of WT and Keap1-KO mice, either left unstimulated or stimulated with anti-CD3/CD28 antibodies, 4 independently performed experiments. The calculated power of this experiment is 0.99 for the stimulated and unstimulated groups. (E) N-fold SLC7A11 expression in human CD4 T cells, either transfected with a control siRNA (Ctrl) or NRF2 siRNA. 3 independently performed experiments were performed. (F) N-fold SLC7A11 mRNA expression in human CD4 T cells treated with 4-OI or vehicle (Ctrl) N = 6, 4 independently performed experiments. A paired Student's t-test was performed comparing the groups of B–F. Data are presented as mean, error bars present ± SEM for all the presented graphs in this figure. Values were considered significant if ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. N represents the number of biological replicates.

Article Snippet: To this, 10 nM NRF2 siRNA (OriGene, Germany; catalogue number SR321100) or a control was added, and transfection was performed according to the manufacturer’s instructions (Neon transfection, ThermoFisher Scientific; MPK10025).

Techniques: Expressing, Flow Cytometry, Control, Comparison, Gene Expression, Microarray, Transfection

Journal: iScience

Article Title: Inflammatory CD4 + T cells can waive NRF2-dependent SLC7A11-mediated cystine uptake by using ASCT1

doi: 10.1016/j.isci.2026.115680

Figure Lengend Snippet: SAS and erastin treatment reduce cystine uptake but not GSH content and cell viability in anti-CD3/CD28 stimulated CD4 + T cells (A) Flow cytometric analysis of cystine uptake measured by BioTracker Cystine-FITC in stimulated CD4 + T cells in the presence of SAS and erastin. Exemplary histogram, showing the cystine uptake after 0 min (blue) and after 30 min (red) of incubation, while the difference presented as ΔMFI was used to describe the cystine uptake. N = 4, 3 independently performed experiments. The calculated power of this experiment is 0.99. (B) Statistical analysis of cystine uptake (represented as MFI) of mouse WT and NRF2-KO CD4 + T cells with and without the addition of SAS and erastin. N = 6, 4 independently performed experiments. (C) Representative histograms of B showing the uptake after 0 min (blue) and 30 min (red), while the difference presented as ΔMFI was used to describe the cystine uptake. (D) Flow cytometric analysis of reactive oxygen species (ROS) (presented as MFI) of CD4 + T cells without and with the addition of SAS and erastin. Histograms of the measurements are shown on the right. The calculated power of this experiment is 0.986. (E) Flow cytometric analysis of intracellular GSH content (presented as ΔMFI) of CD4 + T cells with and without SAS and erastin. Histograms depict an overlay of the FMO (blue) and the GSH staining (red). GSH was determined by the GSH/GSSG Luminescence measurement of CD4 MACS HC PBMCs. The kit provides direct analysis of the supernatants' GSH and GSSG contents, which enables the calculation of the ratios. (F) Lipoperoxidation measurement using BODIPY 581/591 C11 (presented as MFI of the FITC signal) of CD4 + T cells without and with inhibition by SAS and erastin. Histograms of the two signals, i.e., PE and FITC, from BODIPY are shown on the right. The calculated power of this experiment is 0.94. (G) Flow cytometric analysis of Mitotracker (represented as MFI, mitochondrial mass) of CD4 + T cells and TMRM (represented by MFI, mitochondrial membrane potential) of Mitotracker positive cells is shown without and with the addition of SAS and erastin. The calculated power of this experiment is 0.99 for the mitochondrial mass and 1 for the mitochondrial membrane potential. (H) Fluorescent live/dead staining was evaluated by flow cytometry using the combinatory staining of Annexin V and fixable viability dye (FVD) without and with the addition of SAS and erastin. N = 3, 3 independent experiments were performed for the graphs (D) to (G). The statistical evaluation of all the represented graphs was performed using one-way ANOVA multiple comparison. (Data are presented as mean, error bars present ± SEM for all the presented graphs in this figure. Values were considered significant if ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. N represents the number of biological replicates.

Article Snippet: To this, 10 nM NRF2 siRNA (OriGene, Germany; catalogue number SR321100) or a control was added, and transfection was performed according to the manufacturer’s instructions (Neon transfection, ThermoFisher Scientific; MPK10025).

Techniques: Incubation, Staining, Inhibition, Membrane, Flow Cytometry, Comparison

Journal: iScience

Article Title: Inflammatory CD4 + T cells can waive NRF2-dependent SLC7A11-mediated cystine uptake by using ASCT1

doi: 10.1016/j.isci.2026.115680

Figure Lengend Snippet: Characterization of an altered NRF2/cystine pathway in patients with JIA (A) Flow cytometric analysis of SLC7A11 expression in stimulated SF and PB CD4 + T cells. Statistical analysis was performed with N = 7 donors, 7 independently performed experiments. (B) Flow cytometric evaluation of cystine uptake (shown as MFI) by anti-CD3/CD28 stimulated CD4 + SF T cells. N = 6, 6 independently performed experiments. (C) Flow cytometric analysis of intracellular glutathione content (presented as ΔMFI) of stimulated SF and PB CD4 + T cells. N = 9, 9 independently performed experiments. (D) Statistical analysis of MFI of ROS in stimulated SF and PB CD4 + T cells ( N = 4), 4 independently performed experiments. The calculated power of this experiment is 0.85. (E) Lipid peroxidation as assessed by flow cytometric measurement of anti-CD3/CD28 stimulated CD4 + T cells derived from PBMCs and SFMCs. N = 11. (F) Gene set enrichment analysis (GSEA) was performed on differentially expressed genes from SF-derived CD4 + T cells of patients with active JIA compared to PBMC-derived CD4 + T cells from healthy controls. The enrichment plots shown represent driver ferroptosis-related gene sets from KEGG, WikiPathways, and FerrDb V2. ( N = 4). (G) Heat maps showing normalized counts of selected RNAs determined by RNA-seq in JIA CD4 + T cells compared to HC CD4 + T cells. N = 4 in each group. (H) SFMCs and PBMCs were analyzed for ASCT1 expression by quantitative RT-PCR. The calculated power of this experiment is 0.99. (I) Flow cytometric analysis of CD36 expression of stimulated SF and PB CD4 + T cells. N = 5, 5 independently performed experiments. (J) Representative histograms showing BODIPY Ferroptosis staining of anti-CD3/CD28 stimulated HC PBMCs in the absence and presence of ASCT1/2 inhibition by 4-Hydroxy-L-phenylglycin (HPG) and SAS. N = 3, 3 independent experiments. The statistical evaluation of (A), (C), (E), (H), and (I) from the represented graphs was performed using a paired Student’s t-test. One-way ANOVA multiple comparison was used for (B) and (D) of the represented graphs. Data are presented as mean, error bars present ± SEM for all the presented graphs in this figure. Values were considered significant if ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. N represents the number of biological replicates.

Article Snippet: To this, 10 nM NRF2 siRNA (OriGene, Germany; catalogue number SR321100) or a control was added, and transfection was performed according to the manufacturer’s instructions (Neon transfection, ThermoFisher Scientific; MPK10025).

Techniques: Expressing, Derivative Assay, RNA Sequencing, Quantitative RT-PCR, Staining, Inhibition, Comparison

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) TCGA data analysis showing the correlation between SDCBP mRNA and BACH1 mRNA expression in GSE142102 ( n = 226) dataset of TNBC patients (Pearson correlation coefficient r = 0.3245, P < 0.0001). ( B ) TCGA data analysis showing the correlation between SDCBP mRNA and BACH1 mRNA expression in GSE103091 ( n = 238) dataset of TNBC patients (Pearson correlation coefficient r = 0.2120, P < 0.001). ( C ) Western blot showing SDCBP, BACH1, and HO-1 protein expression in MDA-MB-231, MDA-MB-468, Hs578T, MCF-7, and T47D cells. ( D ) The expression levels of SDCBP and BACH1 protein in Fig. EV1C were quantified using densitometry and normalized to the housekeeping protein α-tubulin ( n = 3). ( E ) Real-time qPCR showing SDCBP and BACH1 mRNA expression in MDA-MB-231, MDA-MB-468, Hs578T, MCF-7, and T47D cells ( n = 3). Quantitative data were normalized to β-actin expression. ( F ) Western blot showing SDCBP and HO-1 protein expression in MDA-MB-231 cells transfected with scramble or BACH1 siRNA. ( G ) Left, western blot showing the protein expression of SDCBP in the scramble and in several SDCBP-KO MDA-MB-231 subclones generated using CRISPR-Cas9 system; Right, real-time qPCR showing the SDCBP mRNA expression in scramble and in SDCBP-KO MDA-MB-231 subclones ( n = 3). ( H ) Real-time qPCR showing the mRNA expression of BACH1 in MDA-MB-231 cells, in scramble and in SDCBP-KO MDA-MB-231 subclone#2 and subclone#12 ( n = 3). ( I ) Immunofluorescence staining was used to visualize SDCBP (green color) and BACH1 (red color) in scramble and in SDCBP-KO MDA-MB-231 cells. DAPI (blue color) was used to stain the nucleus ( n = 3); Representative confocal immunofluorescence images are shown. Scale bar = 20 µm. ( J ) Western blot showing BACH1 and HO-1 protein expression in 4T1 cells infected with scramble or adenoviral SDCBP shRNA. ( K ) Real-time qPCR showing the mRNA expression of BACH1-regulated antioxidant genes ( HMOX1, NQO1 , and GLCL ) in 4T1 cells transfected with scramble or SDCBP siRNA ( n = 3); mRNA expression of KEAP1 was the negative control. Data are expressed as the mean ± SEM and analyzed using one-way ANOVA ( D , E , G , H ) or two-way ANOVA ( K ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated.

Article Snippet: Human NRF2 siRNA , Santa Cruz Biotechnology , Cat#sc-37030.

Techniques: Expressing, Western Blot, Transfection, Generated, CRISPR, Immunofluorescence, Staining, Infection, shRNA, Negative Control

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Immunohistochemistry staining against the SDCBP and BACH1 protein in human TNBC-derived tissue microarray sections ( n = 78). Representative images showing the co-expression of SDCBP and BACH1 in the same section. Normal breast cancer tissues were considered as the negative control. Scale bar = 20 µm. ( B ) Pearson correlation coefficient (r = 0.5772, P < 0.0001) between SDCBP and BACH1 expression in ( A ). ( C ) Western blot showing BACH1 and HO-1 protein expression in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector. ( D ) Real-time qPCR showing BACH1 mRNA expression in Hs578T cells transfected with a control vector or a Myc-SDCBP-expressing vector ( n = 3). ( E ) Real-time qPCR showing the mRNA expression of BACH1-regulated antioxidant genes ( HMOX1 and NQO1 ) and BACH1-regulated metastatic genes ( HK2, MMP1 , and CXCR4 ) in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector ( n = 3). ( F ) Western blot showing BACH1 protein expression in MDA-MB-231 infected with lentiviral scramble or SDCBP shRNA. ( G ) Real-time qPCR showing BACH1 mRNA expression in MDA-MB-231 infected with lentiviral scramble or SDCBP shRNA ( n = 3). ( H ) Left, Representative images of immunofluorescence staining to visualize SDCBP ( green color ) and BACH1 ( red color ) expression in MDA-MB-231 cells transfected with a scramble siRNA or SDCBP siRNA. DAPI ( blue color) was used to stain the nucleus ( n = 3); Scale bar = 50 µm. Right, fluorescence levels of SDCBP and BACH1 were quantified based on their spectral densities. ( I ) Real-time qPCR showing the mRNA expression of BACH1-regulated antioxidant genes ( HMOX1 and NQO1 ) and BACH1-regulated metastatic genes ( HK2, MMP1, MMP13 , and CXCR4 ) in MDA-MB-231 cells transfected with scramble siRNA or SDCBP siRNA ( n = 3). ( J ) Western blot showing SDCBP and BACH1 protein expression in scramble control and two SDCBP-KO MDA-MB-231 clones (KO#2, KO#12) generated using CRISPR-Cas9 system ( n = 3). ( K ) Real-time qPCR showing the mRNA expression of BACH1-regulated metastatic genes ( HK2 , MMP1 , CXCR4, GAPDH , and VEGF ) in scramble control and SDCBP-KO MDA-MB-231 cells. ( L ) The reconstitution of SDCBP recovers BACH1 protein expression in SDCBP-KO MDA-MB-231 cells. Western blot showing BACH1 protein expression in scramble control and SDCBP-KO MDA-MB-231 cells transfected with control vector or Myc-SDCBP-expressing vector. The arrows indicate the endogenous (Endo) and exogenous (Exo) SDCBP. Data are expressed as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction ( D , E , G , I ), two-way ANOVA ( H ), or one-way ANOVA ( K ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: Human NRF2 siRNA , Santa Cruz Biotechnology , Cat#sc-37030.

Techniques: Immunohistochemistry, Staining, Derivative Assay, Microarray, Expressing, Negative Control, Western Blot, Transfection, Control, Plasmid Preparation, Infection, shRNA, Immunofluorescence, Fluorescence, Clone Assay, Generated, CRISPR, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Western blot showing BACH1 protein expression in Hs578T cells transfected with Myc-SDCBP-expressing vector with or without BACH1 siRNA. ( B ) Colony formation of Hs578T cells transfected with Myc-SDCBP-expressing vector with or without BACH1 siRNA ( n = 3); See also Fig. . Migration of Hs578T cells transfected with Myc-SDCBP-expressing vector with or without BACH1 siRNA ( n = 3); See also Fig. . ( D ) Western blot showing SDCBP and Flag-BACH1 protein expression in MDA-MB-231 cells transfected with SDCBP siRNA with or without a Flag-BACH1-expressing vector. Cell proliferation of MDA-MB-231 cells transfected with SDCBP siRNA with or without Flag-BACH1-expressing vector ( n = 3). ( F ) Colony formation of MDA-MB-231 cells transfected with SDCBP siRNA with or without Flag-BACH1-expressing vector ( n = 3); See also Fig. . ( G ) Wound closure of scratched MDA-MB-231 cells transfected with SDCBP siRNA with or without a Flag-BACH1-expressing vector ( n = 3); See also Fig. . ( H ) Schematic of various SDCBP mutant constructs generated using the Myc-SDCBP plasmid. ( I ) Left, western blot showing BACH1 protein expression in Hs578T cells transfected with the indicted SDCBP constructs. Right, quantification of BACH1 levels using densitometry ( n = 3). ( J ) Top, schematic of the PDZ1 construct. Bottom, western blot showing BACH1 protein expression in Hs578T cells transfected with Myc-SDCBP or Myc-SDCBP_PDZ1 plasmid. ( K ) Migration of Hs578T cells transfected with Myc-SDCBP, Myc-SDCBP_Δ4, or Myc-SDCBP_PDZ1 plasmid ( n = 3); See also Fig. . ( L ) Colony formation of Hs578T cells transfected with Myc-SDCBP, Myc-SDCBP_Δ4, or Myc-SDCBP_PDZ1 plasmid ( n = 3); See also Fig. . ( M ) Tumor volumes from athymic BALB/c nude mice 6 weeks after mammary fat-pad injection of the scramble control or SDCBP-KO MDA-MB-231 cells (1 × 10 5 cells/mouse; n = 5 or 7 mice/group). ( N ) Tumor weights in Fig. 2M ( n = 7 mice/group); See also Fig. . ( O ) Tumor volumes from athymic BALB/c nude mice 25 days after mammary fat-pad injection of the scramble control, SDCBP-KO MDA-MB-231 cells, and SDCBP-KO MDA-MB-231 cells stably transfected with Flag-SDCBP (1 × 10 5 cells/mouse; n = 5 mice/group); See also Fig. – , . Data are expressed as the mean ± SEM and analyzed using one-way ANOVA ( B , , I , K , L , N ), two-tailed Student’s t test ( , F , O ), or two-way ANOVA ( G , M ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: Human NRF2 siRNA , Santa Cruz Biotechnology , Cat#sc-37030.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Migration, Mutagenesis, Construct, Generated, Injection, Control, Stable Transfection, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Representative images of colony formation in Fig. . ( B ) Representative images of the migrated cells in Fig. . Scale bar = 200 µm. ( C ) Representative images of colony formation in Fig. . ( D ) Representative images of wounding migration in Fig. . Scale bar = 200 µm. ( E ) Cell proliferation of MDA-MB-231 cells transfected with scramble or BACH1 siRNA. Cell proliferation was estimated by an automatic cell counter at the indicated time points ( n = 3). ( F ) Colony formation of MDA-MB-231 cells transfected with scramble or BACH1 siRNA. The clonogenic ability was assessed and quantified based on the absorbance at 600 nm and normalized to the control ( n = 3). ( G ) Migration of MDA-MB-231 cells transfected with scramble or BACH1 siRNA. The number of migrated cells were counted and expressed as percentages ( n = 3). ( H ) Representative images of the migrated cells in Fig. . Scale bar = 500 µm. ( I ) Representative images of colony formation in Fig. . Scale bar = 1000 µm. Data are expressed as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction ( E – G ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated.

Article Snippet: Human NRF2 siRNA , Santa Cruz Biotechnology , Cat#sc-37030.

Techniques: Migration, Transfection, Control, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with SDCBP siRNA with or without MG132 proteasome inhibitor. ( B ) Left, western blot showing BACH1 protein expression in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector in the presence of CHX protein synthesis inhibitor at various time points. Right, quantification of BACH1 protein levels using densitometry ( n = 3). ( C ) Left, western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with scramble or FBXO22 siRNA in the presence of CHX protein synthesis inhibitor at various time points. Right, quantification of BACH1 protein levels using densitometry ( n = 3). ( D ) Western blot showing BACH1, FBXO22, and SDCBP protein expression in MDA-MB-231 cells transfected with scramble or FBXO22 siRNA. ( E ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with HA-FBXO22-expressiong vector with or without Myc-SDCBP-expressing vector. ( F ) In vitro ubiquitylation assay of the recombinant human BACH1 protein mediated by the FBXO22 complex. Active recombinant human UbcH5a protein was used as the E2 ubiquitin-conjugating enzyme for FBXO22 complex-mediated BACH1 degradative polyubiquitylation. ( G ) In vivo ubiquitylation assay showing the decrease in the K48-linked polyubiquitylation of BACH1 by SDCBP overexpression in HEK293 cells transfected with the indicated plasmids. ( H ) In vivo ubiquitylation assay showing the increase in the K48-linked polyubiquitylation of BACH1 by SDCBP KD in MDA-MB-231 cells transfected with the indicated plasmids. ( I ) In vitro ubiquitylation assay showing the inhibitory effect of SDCBP on the polyubiquitylation of BACH1 mediated by the FBXO22 complex. Active recombinant human protein UbcH5a and immunocomplex FBXO22 were added as described above. Recombinant human BACH1 and recombinant human SDCBP proteins were added at ratios of 1:1 (+) and 1:2 (++). Data are expressed as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction ( B , C ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: Human NRF2 siRNA , Santa Cruz Biotechnology , Cat#sc-37030.

Techniques: Western Blot, Expressing, Transfection, Control, Plasmid Preparation, In Vitro, Ubiquitin Assay, Recombinant, Ubiquitin Proteomics, In Vivo, Over Expression, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Left, Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with control vector or Myc-SDCBP-expressing vector in the presence of CHX protein synthesis inhibitor at various time points. Right, quantification of BACH1 protein levels using densitometry ( n = 3). ( B ) Free heme level in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector ( n = 3). ( C ) Free heme level in scramble and in SDCBP - KO MDA-MB-231 cells ( n = 3). ( D ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with control vector or NRF2 (encoded by NFE2L2 )-expressing vector. HO-1 protein expression was considered as the positive control. ( E ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with a control or a HO-1 (encoded by HMOX1 )-expressing vector. ( F ) Western blot showing BACH1 protein expression in Hs578T cells transfected with scramble or HO-1 siRNA. ( G ) Western blot showing BACH1 protein expression in MDA-MB-231 cells transfected with the HO-1 or the catalytic inactive HO-1 mutant (H25A) plasmid. ( H ) Western blot showing BACH1 and SDCBP protein expression in MDA-MB-231 cells transfected with scramble or HOIL1 siRNA. ( I ) Western blot showing endogenous FBXO22 protein expression in several breast cancer cells. ( J ) Immunoprecipitation showing the interaction of BACH1 with FBXO22 in HEK293 cells transfected with the indicated plasmids. An arrow indicates the specific signal for HA-FBXO22. ns: none specific. ( K ) In vivo ubiquitylation assay showing the increase in the polyubiquitylation of BACH1 by FBXO22 overexpression in HEK293 cells transfected with the indicated plasmids. ( L ) In vivo ubiquitylation assay showing the increase in the polyubiquitylation of BACH1 by FBXO22 overexpression in MDA-MB-231 cells transfected with the indicated plasmids. ( M ) In vivo ubiquitylation assay showing the decrease in FBXO22-mediated polyubiquitylation of BACH1 by SDCBP overexpression in HEK293 cells transfected with the indicated plasmids. Data are expressed as the mean ± SEM and analyzed using two-tailed Student’s t test with Welch’s correction ( A – C ). All experiments were repeated at least three times unless otherwise indicated. P values less than 0.05 were considered statistically significant.

Article Snippet: Human NRF2 siRNA , Santa Cruz Biotechnology , Cat#sc-37030.

Techniques: Western Blot, Expressing, Transfection, Control, Plasmid Preparation, Positive Control, Mutagenesis, Immunoprecipitation, In Vivo, Ubiquitin Assay, Over Expression, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Immunoprecipitation showing the interaction of FBXO22 with SDCBP in HEK293 cells transfected with the indicated plasmids. An arrow indicates the specific signal for HA-FBXO22. ( B ) Immunoprecipitation showing the interaction of SDCBP with FBXO22 in HEK293 cells transfected with the indicated plasmids. An arrow indicates the specific signal for HA-FBXO22. ( C ) Co-immunoprecipitation showing the interaction of FBXO22 with BACH1 in HEK293 cells with or without SDCBP after the indicated transfections. ( D ) Schematic of experimental design to investigate the assembly of SCF FBXO22 –BACH1 complex via His Pull-down assay and endogenous IP assay in Fig. D– . ( E ) His-pulldown assay showing the interaction of FBXO22 with SKP1 in HEK293 cells with control vector or Myc-SDCBP-expressing vector after the indicated transfections. See also Appendix Fig. S . ( F ) Western blot showing BACH1, PTEN, and PD-L1 protein expression in scramble and in SDCBP-KO MDA-MB-231 cells. Western blot showing BACH1, PTEN, and PD-L1 protein expression in A549 cells transfected with scramble or SDCBP siRNA. ( H ) Western blot showing BACH1 and PD-L1 protein expression in NCI-H1299 cells transfected with scramble or SDCBP siRNA. ( I ) Western blot showing BACH1, PTEN, and PD-L1 protein expression in Hs578T cells transfected with control vector or Myc-SDCBP-expressing vector. ( J ) In vivo ubiquitylation assay showing the inhibitory effect of SDCBP on SCF FBXO22 -mediated K48-linked polyubiquitylation of BACH1 in HEK293 cells transfected with the indicated plasmids.

Article Snippet: Human NRF2 siRNA , Santa Cruz Biotechnology , Cat#sc-37030.

Techniques: Immunoprecipitation, Transfection, Pull Down Assay, Control, Plasmid Preparation, Expressing, Western Blot, In Vivo, Ubiquitin Assay

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Crosslink immunoprecipitation showing an interaction of FBXO22 with SDCBP in Hs578T cells transfected with Myc-SDCBP-expressing vector or Myc-SDCBP_Δ4-expressing vector. Schematic showing the FBXO22 mutant constructs generated using the HA-FBXO22 plasmid. ( C ) Immunoprecipitation showing SDCBP interactions with FBXO22 and its mutant constructs in HEK293 cells transfected with the indicated plasmids. ns indicates non-specific bands. See also Fig. , . ( D ) His-Pulldown assay showing the effect of SDCBP on SKP1-CUL1-FBXO22 complex formation after the indicated transfections in HEK293 cells. See also Appendix Fig. S . ( E ) His-Pulldown assay showing the effect of SDCBP KO on the SCF FBXO22 –BACH1 complex formation in the scramble control and SDCBP-KO MDA-MB-231 cells transfected with control vector or His-SKP1-expressing vector. See also Appendix Fig. S . ( F ) Immunoprecipitation showing the effect of SDCBP KD on the SCF FBXO22 –BACH1 complex formation in MDA-MB-231 cells transfected with scramble or SDCBP siRNA. ( G ) Immunoprecipitation showing the effect of SDCBP overexpression on the SCF FBXO22 –BACH1 complex formation in Hs578T cells transfected with control vector or Myc-SDCBP-overexpressing vector. ( H ) Immunoprecipitation showing the effect of SDCBP PDZ1 domain on the SCF FBXO22 –BACH1 complex formation in Hs578T cells transfected with a control vector or a Myc-SDCBP-PDZ1-overexpressing vector. .

Article Snippet: Human NRF2 siRNA , Santa Cruz Biotechnology , Cat#sc-37030.

Techniques: Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Construct, Generated, Control, Over Expression

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Real-time qPCR showing the mRNA expression of BACH1-regulated ETC genes ( NDUFA4 , NDUFA4L2 , NDUFC2 , and COX6B2 ) in scramble and SDCBP-KO MDA-MB-231 cells ( n = 3); See also Appendix Fig. S , . Real-time qPCR showing the mRNA expression of NDUFA4 and COX6B2 in scramble, SDCBP-KO MDA-MB-231 cells, and SDCBP-KO MDA-MB-231 cells transfected with SDCBP ( n = 3). ( C ) Western blots showing the expression of mitochondrial proteins NDUFA4 and COX6B2 in MDA-MB-231 cells transfected with SDCBP siRNA in the presence or absence of FLAG-BACH1-expressing vector. ( D ) ChIP-qPCR showing BACH1 enrichments in the promoter regions of NDUFA4 and COX6B2 in the scramble and SDCBP-KO MDA-MB-231 cells. Quantitative data were normalized to IgG binding expression ( n = 3); See also Appendix Fig. S . ( E ) Left, flow cytometry histogram showing the mitochondrial membrane potentials using TMRE (tetramethylrhodamine ethyl ester) staining in MDA-MB-231 cells transfected with a scramble or SDCBP siRNA after the indicated treatments. FCCP (trifluoromethoxy carbonylcyanide phenylhydrazone). Right, quantification of TMRE fluorescence intensity ( n = 3). ( F ) Left, immunofluorescence staining and confocal imaging of the fluorescent signals for TMRE (orange-red color) in the scramble control and SDCBP-KO MDA-MB-231 cells after incubation with TMRE. DAPI (blue color) was used to stain the nucleus ( n = 7); Representative confocal images are shown; scale bars = 20 µm and 5 µm. Right, fluorescent levels of the TMRE were quantified based on their spectral densities. ( G ) Representative images of immunohistochemistry staining against the NDUFA4, BACH1, and SDCBP proteins showing a negative correlation between the expression of NDUFA4 and SDCBP in the same sections of TNBC tumor tissues. Scale bar = 20 µm. ( H ) Pearson correlation coefficients between SDCBP and NDUFA4 protein expression ( n = 64), and between BACH1 and NDUFA4 protein expression ( n = 60) in Fig. 5G. Data are expressed as the mean ± SEM and analyzed using two-way ANOVA ( A , D , F ), two-tailed Student’s t test , or one-way ANOVA ( E ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: Human NRF2 siRNA , Santa Cruz Biotechnology , Cat#sc-37030.

Techniques: Expressing, Transfection, Western Blot, Plasmid Preparation, ChIP-qPCR, Binding Assay, Flow Cytometry, Membrane, Staining, Fluorescence, Immunofluorescence, Imaging, Control, Incubation, Immunohistochemistry, Two Tailed Test

Journal: The EMBO Journal

Article Title: SDCBP/Syntenin-1 stabilizes BACH1 by disassembling the SCF FBXO22 –BACH1 complex in triple-negative breast cancer

doi: 10.1038/s44318-025-00440-1

Figure Lengend Snippet: ( A ) Cell proliferation of MDA-MB-231 cells transfected with a scramble or SDCBP siRNA after treatment with metformin for the indicated periods of time ( n = 3). ( B ) Cell viability of MDA-MB-231 cells transfected with a scramble or SDCBP siRNA after treatment with the indicated concentrations of metformin for 96 h ( n = 5). ( C ) Colony formation for MDA-MB-231 cells transfected with a scramble or SDCBP siRNA after treatment with the indicated concentrations of metformin. Colony numbers were counted and converted to percentages by normalizing with the control groups ( n = 3). ( D – F ) Effect of the indicated treatment on 4T1 tumor growth. Tumor growth was monitored in BALB/c mice bearing 4T1 cells after mammary fat-pad injections. When the average tumor volumes reached 100 mm 3 , the mice ( n = 7 mice/group) were administered with 100 mg/kg metformin (once a day) and/or adenoviral SDCBP shRNA (1 × 10 9 PFU/mice). Black arrows indicate the day of adenoviral SDCBP shRNA injection. Final tumor volume ( E ) and weight ( F ) are shown. ( G ) Immunohistochemistry staining against SDCBP, BACH1, Ki67, NDUFA4, and COX6B2 protein in 4T1 tumors from BALB/c mice in Fig. 6A. Representative images of the IHC staining are shown. Scale bar = 50 µm for low (left) and high (right) magnification. Data are expressed as the mean ± SEM and analyzed using two-way ANOVA ( A – C ), one-way ANOVA ( E ), or two-tailed Student’s t test ( F ). P values less than 0.05 were considered statistically significant. All experiments were repeated at least three times unless otherwise indicated. .

Article Snippet: Human NRF2 siRNA , Santa Cruz Biotechnology , Cat#sc-37030.

Techniques: Transfection, Control, shRNA, Injection, Immunohistochemistry, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: NRF2 activators restrict coronaviruses by targeting a network involving ACE2, TMPRSS2, and XPO1

doi: 10.1101/2025.02.24.639813

Figure Lengend Snippet: Experiments for panels A and B were performed in the Gerold lab (Hannover, Germany) and for panels C-G in the Olagnier lab (Aarhus, Denmark). LDH release assay demonstrated that the compounds were non-toxic to Calu3 cells after 48 h of incubation ( Figure S1 ). A,B . Calu3 cells were pretreated with the compounds (BARD, 0.1 µM; SFN, 10 µM; 4OI, 100 µM) for 24 h, inoculated with SARS-CoV-2/München-1.2/2020/984,p3 (MOI = 0.005) in presence of the compounds for 4 h, followed by removing the viral inoculum and adding fresh medium containing the respective compounds and controls. 48 h p.i., viral genome copies were determined by RT-qPCR. A. supernatants and B. cell lysates. C-G . Calu3 cells were pretreated with the indicated compounds for 48 h (C-F) or 24 h (G), infected with SARS-CoV-2 Wuhan-like early European B.1 lineage (FR-4286) (MOI = 0.01) for 1 h, followed by removal of the inoculum and incubation in fresh medium containing the compounds. Target gene expression and protein levels were measured 24 h p.i. C. Reduction of SARS-CoV-2 spike and nucleocapsid proteins, and XPO1 protein expression, but increase in AKR1B10 and NQO1 levels by 4OI (125 µM) (immunoblot with β-actin as internal reference). D. Marked reduction of viral RNA of diverse SARS-CoV-2 variants of concern by 4OI (125 µM). E,F. NRF2 independence of the anti-SARS-CoV-2 effect of 4OI. Control (transfected with control siRNA) or NRF2 knock-down Calu3 cells (transfected with specific anti-NRF2 siRNA) were infected with SARS-CoV-2 (MOI 0.01) and treated with 4OI (125 µM) or buffer only. E . SARS-CoV-2 RNA (RT-qPCR, cell lysates). F. SARS-CoV-2 spike protein (immunoblot with vinculin as internal reference, cell lysates). n =3, means ±SEM. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Article Snippet: Calu3 cells were transfected in 6-well plates with 80 pmol of human Nrf2 (sc-37030) or control si RNA (sc-37007) (Santa Cruz Bio) diluted in serum and antibiotic free DMEM and using Lipofectamine RNAi Max as per manufacturer’s instructions.

Techniques: Lactate Dehydrogenase Assay, Incubation, Quantitative RT-PCR, Infection, Targeted Gene Expression, Expressing, Western Blot, Control, Transfection, Knockdown

Journal: bioRxiv

Article Title: NRF2 activators restrict coronaviruses by targeting a network involving ACE2, TMPRSS2, and XPO1

doi: 10.1101/2025.02.24.639813

Figure Lengend Snippet: A-F . WT and NRF2 −/- human iPSC-derived ECs were infected with the luciferase-labeled strain hCoV-229E-luc (MOI=0.3) for 4 h and then cultured for 48 h in fresh medium containing the compounds. Luciferase activity, viral M protein RNA levels, host mRNA expression, and mitochondrial ROS levels were measured after 48 h. A,B. Luciferase activity and viral M protein RNA. C. ANPEP mRNA. D. XPO1 mRNA. E. Mitochondrial ROS (flow cytometry). F. HMOX1 mRNA. G-I. Effect of HMOX1 knock-down on 229E-luc infectivity and antiviral activity of the compounds in A549 cells. Infections and treatments were carried out as in A-F, except that WT (transfected with scrambled control siRNA) or siRNA-mediated HMOX1 knock-down A549 cells were used. G. Luciferase activity. H. M protein RNA. I . ANPEP mRNA. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.

Article Snippet: Calu3 cells were transfected in 6-well plates with 80 pmol of human Nrf2 (sc-37030) or control si RNA (sc-37007) (Santa Cruz Bio) diluted in serum and antibiotic free DMEM and using Lipofectamine RNAi Max as per manufacturer’s instructions.

Techniques: Derivative Assay, Infection, Luciferase, Labeling, Cell Culture, Activity Assay, Expressing, Flow Cytometry, Knockdown, Transfection, Control

Journal: International Journal of Biological Sciences

Article Title: Tetramethylpyrazine attenuates sodium arsenite-induced acute kidney injury by improving the autophagic flux blockade via a YAP1-Nrf2-p62-dependent mechanism

doi: 10.7150/ijbs.104107

Figure Lengend Snippet: TMP inhibits YAP1-Nrf2-p62 pathway in rats. (A) AS III-induced p62 activation was verified by increased level of p62 expression in renal tubule, which were inhibited by TMP pretreatment. p62 expression was assessed by immunofluorescence (scale bar =40µm) and IHC staining (scale bar =50µm) in rat kidney tissues. (B) YAP1 expression was assessed by immunofluorescence (scale bar =40µm) and IHC staining (scale bar =50µm). (C) Nrf2 expression was assessed by immunofluorescence (scale bar =40µm) and IHC staining (scale bar =50µm). (D) Semiquantitative analysis of immunofluorescence staining for p62, YAP1, and Nrf2. Figures are representative of 5 rats from each group. Data are represented as mean ± SD (** P <0.01 versus CON. ## P <0.01 versus AS, n =5). (E&F) Western blotting analysis of YAP1, and Nrf2. (** P <0.01 versus CON. ## P <0.01 versus AS, n =3).

Article Snippet: RNA interference siRNA for Nrf2 (human), YAP1 (human) and SQSTM1 (human) was purchased from RiboBio Co., Ltd. (Guangzhou, China).

Techniques: Activation Assay, Expressing, Immunofluorescence, Immunohistochemistry, Staining, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Tetramethylpyrazine attenuates sodium arsenite-induced acute kidney injury by improving the autophagic flux blockade via a YAP1-Nrf2-p62-dependent mechanism

doi: 10.7150/ijbs.104107

Figure Lengend Snippet: TMP attenuates AI-AKI by modulating the Nrf2/p62/Nrf2 feedback loop. (A) Cell viability was measured by CCK8 assay after transfection with Nrf2 and p62 siRNA. (B) Knockdown efficiency was evaluated after successful siRNA transfection for 24 hours. Three different sequences were available for each siRNA, and the two most effective ones were selected for subsequent experiments. NC: Negative Control, PC: Positive Control. (C&D) Western blot for Nrf2 and p62 in HK-2 cells exposed to AS (10μM) or siRNA (50nM) at different time points. ** P <0.01 versus CON, ## P <0.01 versus AS, n =3. (E) After knocking down Nrf2 and p62, the expression of p62 and levels of Nrf2 within the cell nucleus were measured. (F) Quantification of protein levels determined with Western blot. Upper: * P < 0.05, ** P <0.01 compared with CON (or AS and AS+TMP); Lower: ** P <0.01 compared with AS III, # P <0.05, ## P <0.01 versus AS+TMP, n =3. Representative image: Western blot for Nrf2, P62 and LC3B in HK-2 cells exposed to AS (10μM) or TMP (100μM) or siRNA (50nM) at different time points.

Article Snippet: RNA interference siRNA for Nrf2 (human), YAP1 (human) and SQSTM1 (human) was purchased from RiboBio Co., Ltd. (Guangzhou, China).

Techniques: CCK-8 Assay, Transfection, Knockdown, Negative Control, Positive Control, Western Blot, Expressing

Journal: International Journal of Biological Sciences

Article Title: Tetramethylpyrazine attenuates sodium arsenite-induced acute kidney injury by improving the autophagic flux blockade via a YAP1-Nrf2-p62-dependent mechanism

doi: 10.7150/ijbs.104107

Figure Lengend Snippet: TMP alleviates AI-AKI by improving the autophagic flux blockade via YAP1-Nrf2 pathway. (A) Effects on AS III-exposed HK-2 cells after YAP1 agonist. (B) Quantification of protein levels determined with Western blotting. (** P <0.01 versus CON, ## P <0.01 versus AS, n =3). (C&D) Western blotting analysis of YAP1 in cytoplasm and nucleus at different time points under ASIII exposure. (** P <0.01 versus CON, ## P <0.01 versus AS, n =3). (E) Co-immunoprecipitation results of YAP1 and Nrf2. (F) Molecular docking results of YAP1 and Nrf2. Hydrogen-bonding interactions between GLU-100, SER-94, and ARG-87 on the YAP1 protein and LYS-83, GLU-55, and HIS-32 on the Nrf2 protein are their possible binding sites. (G) Immunofluorescence co-localization of YAP1 and Nrf2 at different time points (scale bar = 5µm). (H&I) Western blotting analysis of Nrf2 knockdown on YAP1 expression, and effect of YAP1 knockdown on Nrf2 and LC3B expression after exposure to ASIII at different time points. (* P <0.05, ** P <0.01 compared with AS or AS+TMP, n =3).

Article Snippet: RNA interference siRNA for Nrf2 (human), YAP1 (human) and SQSTM1 (human) was purchased from RiboBio Co., Ltd. (Guangzhou, China).

Techniques: Western Blot, Immunoprecipitation, Binding Assay, Immunofluorescence, Knockdown, Expressing